Abstract
Neutrophils are involved in numerous immunological events. One mechanism of neutrophils to combat pathogens is the formation of neutrophil extracellular traps (NETs). Thereby, neutrophils use DNA fibers to form a meshwork of DNA and histones as well as several antimicrobial components to trap and kill invaders. However, the formation of NETs can lead to pathological conditions triggering among other things (e.g., sepsis or acute lung failure), which is mainly a consequence of the cytotoxic characteristics of accumulated extracellular histones. Interestingly, the carbohydrate polysialic acid represents a naturally occurring antagonist of the cytotoxic properties of extracellular histones. Inspired by polysialylated vesicles, we developed polysialylated nanoparticles. Since sialidases are frequently present in areas of NET formation, we protected the sensitive non-reducing end of these homopolymers. To this end, the terminal sialic acid residue of the non-reducing end was oxidized and directly coupled to nanoparticles. The covalently linked sialidase-resistant polysialic acid chains are still able to neutralize histone-mediated cytotoxicity and to initiate binding of these polysialylated particles to NET filaments. Furthermore, polysialylated fluorescent microspheres can be used as a bioanalytical tool to stain NET fibers. Thus, polySia chains might not only be a useful agent to reduce histone-mediated cytotoxicity but also an anchor to accumulate nanoparticles loaded with active substances in areas of NET formation.
Highlights
More than 10 years ago, a novel defense mechanism of neutrophils was discovered
We already speculate that polysialic acid (polySia) might acts as a molecular anchor to accumulate polysialylated vesicles and immune cells on neutrophil extracellular traps (NETs) [22, 23]
Histones were incubated with increasing concentrations of polySia
Summary
More than 10 years ago, a novel defense mechanism of neutrophils was discovered. Besides the two classical ways—degranulation and phagocytosis—to fight against microbial invasion of pathogens, neutrophils are able to form a DNA-meshwork [1, 2]. Beads were polysialylated by reductive amination adding oxidized colominic acid and 50 mM sodium cyanobrohydride in PBS (pH 7.4). The oxidized colominic acid chains were covalently linked to the particles (fluorescence amine modified latex beads, Sigma-Aldrich) in the presence of sodium cyanoborohydride in PBS (pH 7.4). Samples were incubated with different concentration of polysialylated beads for 1 h at 37°C and mildly washed again with PBS to minimize unspecific interactions. After three times of washing with RPMI, in order to remove the remaining LPS or PMA, cells were incubated with polysialylated beads (325 μg/mL) for 30 min at 37°C. Cells were fixed with 4% paraformaldehyde for 30 min and incubated with 0.5% Triton X-100 (Sigma-Aldrich) for 1 min and washed again with PBS followed by blocking with 2% IgG-free BSA (Carl Roth) at 37°C for 30 min. Significant differences are given: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
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